Stem Cell Based Therapy
Stem cell manipulation for applications in tissue engineering and regenerative medicine has attracted considerable attention.
Embryonic stem cells (EmSC) are stem cells derived from an embryo that are pluripotent, i.e., they are able to differentiate in vitro into endodermal, mesodermal and ectodermal cell types. Embryonic stem (ES) cells are attractive because of their high potential for self-renewal and their pluripotent differentiation capability, but ethical concerns have limited their availability and practical usefulness.
Adult (somatic) stem cells are undifferentiated cells found among differentiated cells in a tissue or organ. Their primary role in vivo is to maintain and repair the tissue in which they are found. Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscles, skin, teeth, gastrointestinal tract, liver, ovarian epithelium, and testis. They reside in a specific local microenvironment of each tissue, known as a stem cell niche, where they may remain quiescent (non-dividing) for long periods of time until they are activated by a normal need for more cells to maintain tissue, or by disease or tissue injury. Examples of adult stem cells include, but not limited to, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells.
Bone marrow consists of a variety of precursor and mature cell types, including hematopoietic cells (the precursors of mature blood cells) and stromal cells (the precursors of a broad spectrum of connective tissue cells), both of which appear to be capable of differentiating into other cell types. Wang, J. S. et al., J. Thorac. Cardiovasc. Surg. 122: 699-705 (2001); Tomita, S. et al., Circulation 100 (Suppl. II): 247-256 (1999); Saito, T. et al., Tissue Eng. 1: 327-43 (1995).
CD34+ cells represent approximately 1% of bone marrow derived nucleated cells. Hematopoietic stem cells (also known as the colony-forming unit of the myeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rare pluripotent cells within the blood-forming organs that are responsible for the continued production of blood cells during life. CD34 antigen also is expressed by immature endothelial cell precursors; mature endothelial cells do not express CD34+. Peichev, M. et al., Blood 95: 952-58 (2000). In vitro, CD34+ cells derived from adult bone marrow give rise to a majority of the granulocyte/macrophage progenitor cells (CFU-GM), some colony-forming units-mixed (CFU-Mix) and a minor population of primitive erythroid progenitor cells (burst forming units, erythrocytes or BFU-E). Yeh, et al., Circulation 108: 2070-73 (2003).
While there is no single cell surface marker exclusively expressed by hematopoietic stem cells, it generally has been accepted that human HSCs have the following antigenic profile: CD 34+, CD59+, Thy1+(CD90), CD38low/−, C-kit−/low and, lin−. CD45 is also a common marker of HSCs, except platelets and red blood cells, which are CD45-. HSCs can generate a variety of cell types, including erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, T lymphocytes, and B lymphocytes. The regulation of hematopoietic stem cells is a complex process involving self-renewal, survival and proliferation, lineage commitment and differentiation and is coordinated by diverse mechanisms including intrinsic cellular programming and external stimuli, such as adhesive interactions with the micro-environmental stroma and the actions of cytokines.
Different paracrine factors are important in causing hematopoietic stem cells to differentiate along particular pathways. Paracrine factors involved in blood cell and lymphocyte formation are called cytokines. Cytokines can be made by several cell types, but they are collected and concentrated by the extracellular matrix of the stromal (mesenchymal) cells at the sites of hematopoiesis. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the multilineage growth factor IL-3 both bind to the heparan sulfate glycosaminoglycan of the bone marrow stroma. The extracellular matrix then presents these factors to the stem cells in concentrations high enough to bind to their receptors.
HSCs reside in the bone marrow but can be forced into the blood, a process termed mobilization. Stem cell mobilization is a process whereby stem cells are stimulated out of the bone marrow into the bloodstream, so they are available for collection from the peripheral blood for future reinfusion. Current mobilization strategies used in the clinic, mainly G-CSF cytokine, are well tolerated but often produce suboptimal number of collected HSCs. HSCs in the bone marrow niche generate energy mainly via anaerobic metabolism and have low levels of ROS, which promotes their self-renewal. Once recruited to the peripheral blood, however, their metabolic state changes, leading to the production of higher levels of ROS, which can induce the cells to differentiate, undergo senescence or lead to apoptosis. (Suda, T. et al, “Metabolic regulation of hematopoietic stem cells in the hypoxic niche,” Cell Devel. 2007; 134(14): 2541-7).
Mobilization and homing are mirror processes depending on an interplay between chemokines, chemokine receptors, intracellular signaling, adhesion molecules and proteases. Homing to the bone marrow is necessary to optimize cell engraftment. The interaction between SDF-1/CXCL12 and its receptor CXCR4 is critical to retain HSCs within the bone marrow. (Suarez-Alvarez, B. et al, “mobilization and homing of hematopoietic stem cells,” Adv. Exp. Med. Biol. 2012; 741: 152-70).
Human umbilical cord blood has long been a focus of attention as an important source of stem cells for transplantation for several reasons, e.g., (1) it contains a higher number of primitive hematopoietic stem cells (HSC) per volume unit, which proliferate more rapidly, than bone marrow; (2) there is a lower risk of rejection after transplantation; (3) transplantation does not require a perfect HLA antigen match (unlike in the case of bone marrow); (4) UC blood has already been successfully used in the treatment of inborn metabolic errors; and (5) there is no need for a new technology for collection and storage of the mononuclear cells from UC blood, since such methods are long established.
Stem cells expressing embryonic molecular markers have been reported from cord blood after removal of hematopoietic cells (including deletion of all leukocyte common antigen CD45 positive cells. (McGuckin, C P, et al, “Production of stem cells with embryonic characteristics from human umbilical cord blood,” Cell Prolif. 2005; 38: 245-55). However, the scarcity of this cell population in cord blood significantly restricts its practical application.
Under certain conditions, an adult differentiated cell can switch its phenotype to that of another mature cell type by transdifferentiation. Transdifferentiation is highly facilitated when the cells are from closely related lineages or are derived from the same embryonic layer. For example, since both the pancreas and liver are endoderm-derived organs, using the appropriate sets of lineage-specific reprogramming transcription factors, hepatocytes can be turned into pancreatic beta cells and vice versa. (Yi, F. et al, “Rejuvenating liver and pancreas through cell transdifferentiation,” Cell Res. 2012; 22(4): 616-619).
In addition, adult somatic cells can be reprogrammed to enter an embryonic stem cell-like state by being forced to express a set of transcription factors, for example, Oct-3/4 (or Pou5f1, the Octamer transcription factor-3/4), the Sox family of transcription factors (e.g., Sox-1, Sox-2, Sox-3, and Sox-15), the Klf family transcription factors (Klf-1, Klf-2, Klf-4, and Klf-5), and the Myc family of transcription factors (e.g., c-Myc, N-Myc, and L-Myc).
For example, human inducible Pluripotent Stem cells (iPSCs) are cells reprogrammed to express transcription factors that express stem cell markers and are capable of generating cells characteristic of all three germ layers (i.e., ectoderm, mesoderm, and endoderm). As originally published by Takahashi et al (Hawkins, K. et al, “Cell signalling pathways underlying iPSc reprogramming,” World J. Stem Cells 2014; 6(5): 620-28, citing Takahashi, K., Yamanaka, S., “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors,” Cell 2006; 126: 663-76), Oct4, Sox2, Klf4 and cMyc were constitutively expressed using genome integrating retroviruses in both mouse and subsequently human fibroblasts, and under ES cell culture conditions were able to induce pluripotency. iPS cells have been successfully generated using episomal plasmids (Id. Citing Yu, J. et al, “Induced pluripotent stem cell lines derived from human somatic cells,” Science 2007; 318: 1917-20), Sendai viruses (Id. Citing Fusaki, N. et al, “Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome,” Proc. Jpn Acad. Ser. B. Phys. Biol. Sci. 2009; 85: 348-62), and transposons (Id. Citing Wang, W. et al, “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 2,” Proc. Natl Acad. Sci. USA 2011; 108: 18283-288) to deliver the reprogramming factors, and even proteins (Id. Citing Zhou, H. et al, “Generation of induced pluripotent stem cells using recombinant proteins,” Cell Stem Cell 2009; 4: 381-84) or small molecules (Hou, P. et al, “Pluripotent stem cells induced from mouse somatic cells by small molecule compounds,” Science 2013; 341: 651-54) alone. The initial need for viral transfection raised concerns about safety with respect to teratogenicity and immunogenicity, and ex vivo transfection of cells may not be stable in the patient. Reprogramming using episomes raises the same concerns. Likewise, chemical reprogramming may not be stable in the patient.
That being said, many diverse cell types have been successfully reprogrammed to pluripotency (Id.). Often, the minimal factors necessary to reprogram a cell depend on the endogenous “stemness” of the starting cell; for example, neural stem cells can be reprogrammed using Oct4 alone since they express high levels of the other factors (Id. Citing Kim, J B, et al, “Oct4-induced pluripotency in adult neural stem cells, “Cell 2009; 136: 411-19).
A “core circuitry” of homeodomain transcription factors, Oct4, Sox2 and Nanog, governs pluripotency in both mouse and human ES cells (Id. Citing Chambers, I., Tomlinson, S R,” The transcriptional foundation of pluripotency,” Development 2009; 136: 2311022). These transcription factors are expressed both in vivo in the inner cell mass of the blastocyst and in vitro in pluripotent cells. Their close interaction facilitates the precise regulation of the core circuitry necessary to maintain the pluripotent state; for instance Oct4 overexpression leads to endoderm and mesoderm differentiation, whereas blockade of Oct4 induces trophoblast differentiation (Id. Citing Niwa, H. et al, “Quantitative expression of Oct3/4 defines differentiation, dedifferentiation or self-renewal of ES cells,” Nat. Genet. 2000; 24: 372-76). Low levels of Oct4 result in upregulation of Nanog, whereas higher levels of Oct4 result in downregulation of Nanog (Id. Citing Loh, Y H, et al, “The Oct4 and Nanog transcriptin network regulates pluripotency in mouse embryonic stem cells,” Nat. Genet. 2006′ 38: 431-440). Similarly, small increases in Sox2 expression or ablation of Sox 2 expression both induce multilineage differentiation (Id. Citing Klopp, J L et al, “Small increases in the level of Sox2 trigger the differentiation of mouse embryonic stem cells,” Stem Cells 2008; 26: 903-11). All 3 factors have been shown to regulate the expression of each other as well as themselves. (Id. Citing Boyer, L A et al, “Core transcriptional regulatory circuitry in human embryonic stem cells,” Cell 2005; 122: 947-56; Loh, Y H, et al, “The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells,” Nat. Genet. 2006′ 38: 431-440; Pan, G. et al, “A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal,” FASEB J. 2006; 20: 1730-32).
Cell Signalling Pathways Underlying iPSc Reprogramming
Induced pluripotent stem cell reprogramming consists of three phases: initiation, maturation, and stabilization. Hawkins, K. et al, “Cell signalling pathways underlying iPSc reprogramming,” World J. Stem Cells 2014; 6(5): 620-28), citing Samavarchi-Tehrani et al (36).
The initiation phase is characterized by somatic genes being switched off by methylation, an increase in cell proliferation, a metabolic switch from oxidative phosphorylation to glycolysis, reactivation of teleomerase activity and a mesenchymal-to-epithelial transition (MET)(Id. Citing David, L, and Polo, J M, “Phases of reprogramming,” Stem Cell Res. 2014; 12: 754-61), which involves the loss of mesenchymal characteristics, such as motility, and the acquisition of epithelial characteristics, such as cell polarity and expression of E-Cadherin (Id. Citing Redmer, T et al, “E-cadherin is crucial for embryonic stemcell pluripotency and can replace OCT4 during somatic cell reprogramming,” EMBO Rep. 2011; 12: 720-26). Mechanistically, Sox2 suppresses expression of Snail, an EMT inducer (Id. Citing Liu, X et al, “Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming,” Nat. Cell Biol. 2013; 15: 829-38), and Klf4 induces E-cadherin expression, thus promoting MET (Id. Citing Li, R et al., “A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts,” Cell Stem Cell 2010; 7: 51-63). TGFβ inhibition can enhance the initiation stage of both mouse (Id. Citing Maherali, N et al, “Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc,” Curr. Biol. 2009; 19: 1718-23; Shi, Y. et al, “A combined chemical and genetic approach for the generation of induced pluripotent stem cells,” Cell Stem Cell 2008; 2: 525-28) and human somatic cell reprogramming (Id. Citing Lin, T., et al, “A chemical platform for improved induction of human iPSCs,” Nat. Methods 2009; 6: 805-808), showing that addition of recombinant TGFβ abrogates iPS cell formation, likely due to the EMT-inducing action of TGFβ signaling, which then prevents MET. TGFβ inhibitors promote Nanog expression, and mitogen-activated protein kinase (MAPK) signalling, activated by TGFβ, further induces the expression of mesodermal genes. (Id. Citing Thierry, J P, Sleeman, J P, “Complex networks orchestrate epitheial-mesenchymal transitions,” Nat. Rev. Mol. Cell Bio. 2006; 7: 131-42). Inhibitors of MAPK signalling have therefore been used in combination with TGFβ inhibitors to promote MET (Id. Citing Lin, T., et al, “A chemical platform for improved induction of human iPSCs,” Nat. Methods 2009; 6: 805-808).
Bone morphogenetic protein (BMP) signaling also plays an important role in the initiation stage of mouse iPS cell reprogramming by promoting MET via upregulation of epithelial genes, such as E-cadherin, occludin and epithelial cell adhesion molecule (Id. Citing Samavarchi-Tehrani, P. et al, “Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition In the initiation of somatic cell reprogramming,” Cell Stem Cell 2010; 7: 64-77). However, constitutive BMP activation prevents human somatic cell reprogramming.
Fibroblast growth factor (FGF) signaling has also been implicated at the initiation stage (Id. Citing Jiao, J. et al, “Promoting reprogramming by FGF2 reveals that the extracellular matrix is a barrier for reprogramming fibroblasts to pluripotency,” Stem Cells 2013; 31: 729-740). It has been shown that FGF2 promotes the early stages of reprogramming through accelerating cell proliferation, facilitating MET and eliminating extracellular collagens. In addition to an increased proliferation rate, the minority of cells that undergo successful reprogramming also exhibit resistance to apoptosis and senescence by transgene expression (Id. Citing Papp, B, “Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape,” Cell Res. 2011; 21: 486-501).
The initiation phase is also characterized by a metabolic switch from oxidative phosphorylation to glycolysis (Id. Citing Panopoulos, A D et al, “The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming,” Cell Res. 2012; 22: 168-77), which involves PI3K/AKT signaling (Id. Citing Zhu, S. et al, “Reprogramming of human primary somatic cells by OCT4 and chemical compounds,” Cell Stem Cell 2010; 7: 651-55; Chen, M. et al, “Promotion of the induction of cell pluripotency through metabolic remodeling by thyroid hormone triiodothyronine-activated PI3K/AKT signal pathway,” Biomaterials 2012; 33: 5514-23).
During the maturation phase, epigenetic changes occur allowing expression of the first pluripotency-associated genes (Id. Citing David, L, and Polo, J M, “Phases of reprogramming,” Stem Cell Res. 2014; 12: 754-61), which include Fbxo15, Sal4, Oct4, Nanog and Esrrb. LIF/STAT3 signaling is required for the maturation phase of mouse iPS cell reprogramming (Id. Citing Tang, Y and Tian, X C, “JAK-STAT3 and somatic cell reprogramming,” JAK-STAT 2013; 2: e24935). Wnt signaling also enhances the maturation phase of mouse somatic cell reprogramming, whereby exogenous stimulation of the pathway using Wnt3a after induction of reprogramming enhances formation of Nanog positive colonies (id. Citing Ho, R, et al, “Stage-specific regulation of reprogramming to induced pluripotent stem cells by Wnt signaling and T cell factor proteins,” Cell Rep. 2013; 3: 2113-26).
The stabilization phase is characterized by transgene independence; therefore, only cells that have activated endogenous pluripotency gene expression are able to maintain pluripotency at this late stage.
Platelets
Platelets (thrombocytes), anucleate discoid-shaped cell fragments generated from large (50 to 100 μm in diameter) multinucleated (up to 128 N) megakaryocytes (MK), play a central role in hemostasis (meaning the stoppage of blood loss at sites of vascular injury) and vascular repair. Principles of Tissue Engineering, 4th Ed., Robert Lanza, Robert Langer, Joseph Vacanti, Eds, Elsevier, Inc.: New York, 2014 at 1047-1048. They represent about 3×1011 cells/liter in peripheral blood, i.e., second only to those of RBCs. Platelets have a short life span, lasting only 7-9 days in the circulation.
Platelet Function
Primary hemostasis is achieved through a synergistic network of receptor/ligand interactions that result in platelet adhesion and simultaneous platelet activation, platelet secretion to activate nearby platelets, platelet aggregation, and ultimately formation of a platelet plug and generation of a surface amenable to assembly of coagulation factor complexes. Haley, K M et al, “Neonatal platelets: mediators of primary hemostasis in the developing hemostatic system,” Pediatr. Res. 2014; 76(3): 230-37.
Platelet Adhesion.
Following vascular injury and the attendant endothelial damage, platelet adhesion, initiating the process of primary hemostasis, is mediated through receptor/ligand interactions in a step-wise fashion. Id. Extracellular von Willebrand factor (VWF)-platelet glycoprotein (GP) Ib binding mediates initial platelet recruitment to the injured area. Id. Platelet GPVI interacts with fibrillary collagen and platelet β1 integrin interacts with laminin, collagen and fibronectin, allowing for firm adhesion of platelets to the exposed extracellular matrix. Id.
Platelet Activation.
Following platelet adhesion, a series of downstream signaling events results in an increase in intracellular calcium and subsequent platelet activation marked by exposure of negatively-charged phosphatidylserine (PS) on the platelet membrane surface, allowing for the assembly of coagulation factors; platelet alpha and delta granule secretion, resulting in the release of ADP, calcium, serotonin, VWF, coagulation factors V and VIII, and fibrinogen; platelet membrane GPIIb/IIIa integrin conversion to a high affinity state for VWF and fibrinogen binding; thromboxane A2 generation through arachidonic acid metabolism; and cytoskeletal reorganization to increase the surface area of spread platelets. Id.
Platelet Aggregation.
A key step for the development of a stable platelet aggregate is the conversion of the GPIIb/IIIa receptor into its high affinity conformation. Id. This allows for stable interactions between the receptor and fibrinogen, VWF, and fibronectin. Platelets aggregate together, forming a platelet plug, the end product of primary hemostasis.
Platelet Markers
Markers that Appear on the Platelet Surface Before Activation.
Platelet surface markers, which appear on the platelet surface before activation, include CD41 (GP IIb/IIIa), CD42a (GPIX), CD42b (GPIb), and CD61 (avb3, vitronectin receptor).
Integrin alpha chain 2b (CD41) is a heterodimeric integral membrane protein that undergoes post-translational modifications that result in two polypeptide chains linked by a disulfide bond. CD41 is expressed on platelets and megakaryocytes and on early embryonic hematopoietic stem cells. A CD41/CD61 complex formed by the integrin alpha chain associated with a beta 3 chain (CD61) Integrin αIIIbβ3 is a receptor for fibronectin, fibrinogen, von Willebrand factor, vitronectin and thrombospondin, and plays an important role in coagulation. The GPIIb/IIIa receptor (integrin αIIbβ3) is one of the most abundant cell surface receptors (=80 000 per platelet) [Wagner C L, Mascelli M A, Neblock D S, Weisman H F, Coller B S, Jordan R E. Analysis of GPIIb/IIIa receptor number by quantitation of 7E3 binding to human platelets. Blood. 1996; 88:907-914], which represents about 15% of total surface protein. [Jennings, L K, Phillips, D R, “Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-Ill complex. J Biol Chem. 1982; 257:10458-10466]. On quiescent platelets, this receptor exhibits minimal binding affinity for von Willebrand factor and plasma fibrinogen. [French, D L, Seligsohn, U, “Platelet Glycoprotein IIb/IIIa receptors and Glanzmann's throbasthenia,” Arteriosclerosis, thrombosis and Vascular Biology 2000: 20: 607-610].
CD42a-d complex is a receptor for von Willebrand factor and thrombin. CD42a is also called platelet glycoprotein GPIX, GP9a. CD42b is also called platelet GPIb alpha, or glycoprotein 1b-alpha.
Markers which Appear on the Platelet Surface During Activation.
Examples of markers which appear on the platelet surface during activation include activated IIb/IIIa, CD62P (P-selectin), CD31 (PECAM) and CD63.
In an activated state, “inside-out” signal transduction mechanisms [Shattil S J, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood. 1998; 91:2645-2657] trigger a conformational change in the GPIIb/IIIa receptor (integrin αIIbβ33) to a high-affinity ligand-binding state that is competent to bind adhesive glycoproteins and form a platelet plug.
P-selectin mediates the initial adhesion of activated platelets to monocytes and neutrophils via the P-selectin glycoprotein ligand-1 (PSGL-1) counterreceptor on the leukocyte surface. [Michelson, A D and Furman, M I, “Markers of Platelet Activation and Granule Secretion,” in Contemporary Cardiology: Platelet Function: Assessment, Diagnosis and Treatment, M. Quinn and D. Fitzgerald, Eds, Humana Press, Towaco N.J.: 2005]. It is a component of the α granule membrane of resting platelets that is only expressed on the platelet surface membrane after a granule secretion. Id. In vivo, circulating degranulated platelets rapidly lose their surface P-selectin but continue to circulate and function. Id. Soluble P-selectin in plasma may be of endothelial cell origin. Id.
Soluble CD40 ligand (sCD40L, CD154) is a plasma marker of in vivo platelet activation. Id. Release of sCD40L by activated platelets is the predominant source of plasma sCD40L; the mechanism of sCD40L release is proteolysis of platelet surface CD40L. Id. Accurate measurement of in vivo circulating sCD40L requires assay in plasma rather than serum. Id.
Lysosomal Activated Membrane Protein (CD63) is a cell surface glycoprotein that is known to complex with integrins. It may function as a blood platelet activation marker.
Platelet surface P-selectin (CD62P) is a component of the α-granule membrane of resting platelets that is only expressed on the platelet surface membrane after a granule secretion.
Megakaryopoiesis and Thrombopoiesis
Megakaryocytes (MK), the precursor of platelets, provide a constant source of platelets to the blood system, and are themselves produced through the process of megakaryopoiesis. As with RBCs MKs are generated through the initial differentiation of hematopoietic stem cells (HSCs) into common myeloid progenitors (CMPs). (Kaushansky, K., “Historical Review: megakaryopoiesis and thrombopoiesis,” Blood 2008; 111(3): 981-86). Progressive commitment of CMPs to the megakaryocyte lineage is principally regulated by thrombopoietin (TPO). The committed megakaryocyte progenitor cells, colony forming units-megakaryocyte (CFU-MK), proliferate and differentiate into megakaryocytes. Id. The maturation of a megakaryocyte involves an increase in expression of the cell surface markers GPIIb/IIIa (also known as CD41 or αIIb/βIII integrin receptor) and GPIb/GPIX/GPV receptors, and a substantial increase in cell mass, which results in cytosolic accumulation of a granules, dense bodies, and platelet-associated proteins like von Willebrand factor (vWF) and platelet factor-4. Id. Several rounds of endomitosis lead to polyploidization and cells with up to 128N in DNA content. Id. Once polyploid MKs are produced, cellular processes on the MK body called ‘protoplatelets’ begin to appear, with their eventual fragmentation and release, resulting in the generation of platelets. Id.
HSCs from peripheral blood (PB), bone marrow (BM) and CB are also capable of producing megakaryocytes and functional platelets. (See Norol, F et al, Effects of cytokines on platelet production from blood and marrow CD34+ cells. Blood 1998; 91(3); 830-43; Bruno, S. et al, In vitro and in vivo megakaryocyte differentiation of fresh and ex-vivo expanded cord blood cells; rapid and transient megakaryocyte reconstitution. Haematologica 2003; 88(4): 379-87; Ungerer, M et al, Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment; Cir. Res. 2004; 95(5): e36-44).
Cellular Origins of Megakaryopoiesis
Two colony morphologies that contain exclusively megakaryocytes have been described in semisolid media. [Kaushansky, K., “Historical review: megakaryopoiesis and thrombopoiesis, Blood 2001; 111(3): 981-86]. The colony-forming unit-megakaryocyte (CFU-MK) is a cell that develops into a simple colony containing from 3 to 50 mature megakaryocytes; larger, more complex colonies that include satellite collections of megakaryocytes and contain up to several hundred cells are derived from the burst-forming unit-megakaryocyte (BFU-MK). Id. Because of the difference in their proliferative potential and by analogy to erythroid progenitors, BFU-MK and CFU-MK are thought to represent the primitive and mature progenitors restricted to the lineage, respectively. Id. Like their erythroid counterparts, the cytokine requirements for CFU-MK are simple; thrombopoietin stimulates the growth of 75% of all CFU-MK, with interleukin (IL)-3 being required along with thrombopoietin for the remainder. IL-3 or steel factor (SF) is required along with thrombopoietin for more complex, larger MK colony formation from primitive progenitor cells.
Megakaryocytes also arise in clonal colonies containing cells of one or more additional hematopoietic lineages. The most primitive in vitro colony-forming cell is termed a colony-forming unit-granuloycte-erythrocyte-monocyte-megakaryocte (CFU-GEMM), mixed progenitor colony (CFU-Mix), or common myeloid progenitor (CMP; Id. Citing Akashi K, Traver D, Miyamoto T, Weissman I L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000; 404:193-19719), and colonies derived from this cell often contain several megakaryocytes. A derivative of the CMP is the mixed MK/erythroid progenitor cell (MEP; Id. Citing Nakorn T N, Miyamoto T, Weissman I L. Characterization of mouse clonogenic megakaryocyte progenitors. Proc Natl Acad Sci USA. 2003; 100:205-210). Before their purification, the existence of an MEP was postulated based on the many common features of cells of the erythroid and megakaryocytic lineage, including the expression of several common transcription factors (SCL, GATA1, GATA2, NF-E2), cell surface molecules (TER119), and cytokine receptors (for IL-3, SF, erythropoietin, and thrombopoietin), and the finding that most erythroid and MK leukemia cell lines display, or can be induced to display, features of the alternate lineage. (Id. Citing McDonald T P, Sullivan P S. Megakaryocytic and erythrocytic cell lines share a common precursor cell. Exp Hematol. 1993; 21:1316-1320; Nakahata T, Okumura N. Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers. Leuk Lymphoma. 1994; 13:401-409) Moreover, the cytokines most responsible for development of these two lineages, erythropoietin and thrombopoietin, the two most closely related proteins in the hematopoietic cytokine family (Id. Citing Lok S, Kaushansky K, Holly R D, et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature. 1994; 369:565-568), display synergy in stimulating the growth of progenitors of both lineages. (Id. Citing Broudy V C, Lin N L, Kaushansky K. Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood. 1995; 85:1719-1726).
The transcription factors expressed by megakaryocytic progenitors that allow for their commitment to the lineage include GATA1, and FOG29. GATA1 is an X-linked gene encoding a 50 kDa zinc finger DNA binding protein. (Id. Citing Martin D I, Zon L I, Mutter G, Orkin S H. Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature. 1990; 344:444-447). Genetic elimination of the transcription factor established the critical role of this transcription factor in hematopoiesis; the GATA1−/− condition is embryonic lethal due to failure of erythropoiesis (Id. Citing Pevny L, Simon M C, Robertson E, et al. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991; 349:257-260), and megakaryocyte-specific elimination of GATA1 leads to severe thrombocytopenia due to dysmegakaryopoiesis. (Id. Citing Shivdasani R A, Fujiwara Y, McDevitt M A, Orkin S H. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 1997; 16:3965-3973). GATA1 acts in concert with friend of GATA (FOG29), another protein that affects transcription without binding to DNA,
The ets family of transcription factors includes approximately 30 members that bind to a purine box sequence, and consists of proteins that interact in both positive and antagonistic ways. For example, PU.1, initially termed Spi-1 based on its association with spleen focus-forming virus products, blocks erythroid differentiation, although it supports megakaryocyte development. (Id. Citing Doubeikovski A, Uzan G, Doubeikovski Z, et al. Thrombopoietin-induced expression of the glycoprotein IIb gene involves the transcription factor PU. 1/Spi-1 in UT7-Mpl cells. J Biol Chem. 1997; 272:24300-24307). Moreover, the ets factor Fli-1 is essential for megakaryopoiesis (Id. Citing Athanasiou M, Clausen P A, Mavrothalassitis G J, Zhang X K, Watson D K, Blair D G. Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype. Cell Growth Differ. 1996; 7:1525-1534), and mutations in the genetic region of the transcription factor are associated with congenital thrombocytopenia in humans. (Id. Citing Hart A, Melet F, Grossfeld P, et al. Fli-1 is required for murine vascular and megakaryocytic development and is hemizygously deleted in patients with thrombocytopenia. Immunity. 2000; 13:167-177).
Thrombopoiesis
Thrombopoiesis is the process of formation of thrombocytes (platelets). On a molecular level, thrombopoiesis is a highly coordinate process, with sophisticated reorganization of membrane and microtubules and precise distribution of granules and organelles. Platelets form by fragmentation of mature megakaryocyte membrane pseudopodial projections termed proplatelets (Kaushansky citing Patel S R, Hartwig J H, Italiano J E., Jr The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest. 2005; 115:3348-3354), in a process that consumes nearly the entire cytoplasmic complement of membranes, organelles, granules, and soluble macromolecules. It has been estimated that each megakaryocyte gives rise to 1000 to 3000 platelets (Id. Citing Stenberg P E, Levin J. Mechanisms of platelet production. Blood Cells. 1989; 15:23-47) before the residual nuclear material is eliminated by macrophage-mediated phagocytosis. (Id. Citing Radley J M, Haller C J. Fate of senescent megakaryocytes in the bone marrow. Br J Haematol. 1983; 53:277-287). This process involves massive reorganization of megakaryocyte membranes and cytoskeletal components, including actin and tubulin, during a highly active, motile process in which the termini of the process branch and issue platelets. (Id. Citing Italiano J E, Jr, Lecine P, Shivdasani R A, Hartwig J H. Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol. 1999; 147:1299-1312) Localized apoptosis may play a role in initiating the final stages of platelet formation, (Id. Citing Li J, Kuter D J. The end is just the beginning: megakaryocyte apoptosis and platelet release. Int J Hematol. 2001; 74:365-374; De Botton S, Sabri S, Daugas E, et al. Platelet formation is the consequence of caspase activation within megakaryocytes. Blood. 2002; 100:1310-1317) potentially by allowing the issuing of proplatelet processes from the constraints of the actin cytoskeleton. During the final stages of proplatelet maturation, cytoplasmic organelles and secretory granules traffic to the distal tips of proplatelet processes and are trapped there. (Id. Citing Richardson J L, Shivdasani R A, Boers C, Hartwig J H, Italiano J E., Jr Mechanisms of organelle transport and capture along proplatelets during platelet production. Blood. 2005; 106:4066-4075) Microtubules sliding over one another are the engine that drives the elongation of proplatelet processes and organelle transportation. (Id. Citing Patel S R, Richardson J L, Schulze H, et al. Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes. Blood. 2005; 106:4076-4085). While thrombopoietin is the primary regulator of thrombopoiesis, little is known about what determines the size of mature platelets or how the mechanism of platelet formation is affected by the transcription factor GATA1, the glycoprotein Ib/IX complex, the Wiskott Aldrich syndrome protein (WASP), and platelet myosin, as defects in each of these genes leads to unusually large or small platelets. (Id. Citing Geddis A E, Kaushansky K. Inherited thrombocytopenias: toward a molecular understanding of disorders of platelet production. Curr Opin Pediatr. 2004; 16:15-22). Despite the importance of thrombopoietin for the generation of fully mature megakaryocytes from which platelets arise, elimination of the cytokine during the final stages of platelet formation is not detrimental (Id. Citing Choi E S, Nichol J L, Hokom M M, Hornkohl A C, Hunt P. Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood. 1995; 85:402-413).
Umbilical Cord
Two types of umbilical stem cells can be found, namely hematopoietic stem cells (UC-HS) and mesenchymal stem cells, which in turn can be found in umbilical cord blood (UC-MS) or in Wharton's jelly (UC-MM).
Umbilical cord (UC) vessels and the surrounding mesenchyma (including the connective tissue known as Wharton's jelly) derive from the embryonic and/or extraembryonic mesodermis. Thus, these tissues, as well as the primitive germ cells, are differentiated from the proximal epiblast, at the time of formation of the primitive line of the embryo, containing MSC and even some cells with pluripotent potential. The UC matrix material is speculated to be derived from a primitive mesenchyma, which is in a transition state towards the adult bone marrow mesenchyma (Mihu, C. et al., 2008, Romanian Journal of Morphology and Embryology, 2008, 49(4):441-446).
The blood from the placenta and the umbilical cord, which contains all the normal elements of blood—red blood cells, white blood cells, platelets and plasma—is relatively easy to collect in usual blood donation bags, which contain anticoagulant substances. Mononuclear cells then are separated by density gradient centrifugation. In Ficoll-Paque density gradient centrifugation, anticoagulant-treated and diluted cord blood is layered on the Ficoll-Paque solution and centrifuged. During centrifugation, erythrocytes and granulocytes sediment to the bottom layer. Cord blood mononuclear cells and other slowly sedimenting particles with low density (e.g., platelets) are retained at the interface between the plasma and Ficoll-Paque, where they can be collected. (Jaatinen, T., Laine, J. “Isolation of Mononuclear Cells from Human Cord Blood by Ficoll-Paque Density Gradient,” Unit 2A, Curr. Protocols in Stem Cell Biol., DOI: 10.1002/9780470151808.sc02a01 s1). Exposure of the cells at the interface to platelet growth factors has the potential to affect the functional properties of such cells (Aghideh, A N et al, “Platelet growth factors suppress ex vivo expansion and enhance differentiation of umbilical cord blood CD133+ stem cells to megakaryocyte progenitor cells,” Growth Factors 2010; 28(6): 409-16, Citing Voss, et al, “Flow cytometric detection of platelet activation in patients undergoing diagnostic and interventional coronary angiography, “Platelets 1996; 7: 237-41 1996; Gutensohn, K. et al, “Flow cytometric analysis of platelet membrane antigens during and after continuous flow plateletpheresis,” Transfusion 1997: 37: 809-15; Gutensohn, K. “Alteration of platelet-associated membrane glycoproteins during extracorporeal apheresis of peripheral blood progenitor cells,” J. Hematother. 1997; 6: 315-21; Stroncek, et al., “/composition of peripheral blood progenitor cell components collected from a healthy donors [sic],” Transfusion 1997; 37: 411-17; Stroncek, D F et al, “Collection of two peripheral blood stem cell concentrates from healthy donors,” Transfus. Med. 1999; 9: 37-50; Bruserud, O et al, “Autologous stem cell transplantation as post-remission therapy in adult acute myelogenous leukemia: Does platelet contamination of peripheral blood mobilized stem cell grafts influence the risk of leukemia relapse?, J. Hematother. Stem cell Res. 2000; 9: 433-43; Saigo, K., et al., “RANTES and p-Selectin in peripheral blood stem,” Ther. Apher. Dial. 2001; 5: 517-18).
The mononuclear cell fraction includes two stem cell populations: (1) hematopoietic stem cells (HSC), which express certain characteristic markers (CD34, CD133); and (2) mesenchymal stem cells (MSC) that are capable of adhering to a culture surface under certain conditions (e.g., modified McCoy medium and lining of vessels with Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS)). (Munn, D. et al., Science, 1998, 281: 1191-1193; Munn, D. et al., J Exp Med, 1999, 189: 1363-1372). Umbilical cord blood MSCs (UC-MS) can produce cytokines, which facilitate grafting in the donor and in vitro HSC survival compared to bone marrow MSC. (Zhang, X et al., Biochem Biophys Res Commun, 2006, 351: 853-859).
MSCs from the umbilical cord matrix (UC-MM) are obtained by different culture methods depending on the source of cells, e.g., MSCs from the connective matrix, from subendothelial cells from the umbilical vein or even from whole umbilical cord explant. They are generally well cultured in DMEM medium, supplemented with various nutritional and growth factors; in certain cases prior treatment of vessels with hyaluronic acid has proved beneficial (Baban, B. et al., J Reprod Immunol, 2004, 61: 67-77).
Human umbilical cord blood (HUCB) is rich in hematopoietic progenitor cells, as measured in standard clonogenic assays for burst-forming units and granulocyte-macrophage colony-forming units. (Cicuttini, F M and Boyd A W, “Hematopoietic and lymphoid progenitor cells in human umbilical cord blood,” Devel. Immunol. 4: 1-11 (1994), citing Broxmeyer, H E et al, “Human umbilical cord blood as a potential source of transplantable hemopoietic stem/progenitor cells,” Proc. Natl Acad. Sci. USA (1989) 86: 3828-3719).
In vitro culture of human umbilical cord blood has demonstrated multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells (Id. citing Leary, A G et al, “Single cell origin of multi-lineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors ar stochastic processes,” J. Clin. Invest. (1984); 74: 2193-97). A proportion of colonies also contains progenitors that form secondary colonies when replated in a secondary agar culture, suggesting that the colony arises from a single cell with limited self-renewal properties. The frequency of cord blood progenitors (number of colonies formed/number of cells plated) equals or exceeds that of marrow and greatly surpasses that of adult blood. Progenitor cells from HUCB can be maintained for several weeks in long-term liquid culture systems, suggesting their production from more primitive cells (Id. citing Salahuddin, S Z et al, “Long term suspension cultures of human cord blood myeloid cells,” 1981; Blood 58: 931-38; Smith, S & Broxmeyer, H E; “The influence of oxygen tension on the long-term growth in vitro of hemopoietic progenitor cells from human cord blood,” Brit. J. Haematol. (1986): 63: 29-34).
Purification of highly purified CD34+ progenitor cells from HUCB by immunodepletion followed by positive FACS sorting resulted in >100 fold enrichment of colony-forming cells (CFC). Id. Cord blood progenitor cells were shown to be skewed to very early cells in that cord blood CD34+ cells grown in the presence of stem cell factor (SCF) and optimal growth factors resulted in 50-80% of mixed colonies (CFU-GEMM), suggesting that the stem/progenitor cell pool in cord blood is weighted toward very early progenitor cells. Id.
Cord Blood B Cells
Human umbilical cord blood has been shown to be enriched for pre-B and B cells compared to adult peripheral blood. Id. The mean frequency of pre-B cells has been shown to be 0.7% of total lymphocytes in cord blood compared to 0.2% in adult blood (Id. citing Okino, F, “Pre-B cells and B lymphocytes in human cord blood and adult peripheral blood,” Acta Paediatr. Jpn (1987): 29: 195-201). The mean relative frequency of B lymphocytes in cord blood is also higher, being 11.4% of total lymphocytes compared to 5.4% in adult blood (Id). In terms of absolute numbers of preB cells, cord blood contains 10 times the number in adult blood. Id.
The antigens CD1C, CD38, CD5 and CD23 are highly expressed on cord blood B cells, but are normally expressed on only a small percentage of circulating B cells in normal adults. Id. It has been suggested that whereas neonatal B cells are probably functionally naïve, their inherent potential for stimulation, which approaches that of adult B cells, can be realized as long as sufficiently strong T-cell help is available. Id.
Cord Blood T Cells
Generally cord blood T cells have a relative absence of helper activity (Id. citing Anderson, U et al., Evidence for the ontogenic precedence of suppressor T cell functions in the human neonate,” Eur. Immunol. 1983; 13: 6-13). The percentage of lymphocytes expressing CD2 (a surface antigen of the human T-lymphocyte lineage that is expressed on all peripheral blood T cells), CD3 (T lymphocyte marker) and CD8 (marker for T cells with suppressor and cytotoxic activity) is lower in cord blood than in adult blood. Id. However, due to the increased white cell count in cord blood, the absolute numbers of CD2+ and CD8+ cells are comparable (Id. citing Gerli, R et al, Activation of cord T lymphocytes. I. Evidence for a defective T cell mitogens through the CD molecule,” J. Immunol. 1989; 142: 2583-89). In contrast, the percentages of CD4+ cells (helper/inducer T cells) in cord blood and adult peripheral blood are similar, although the absolute numbers of CD4+ cells are higher in cord blood. Id. Nevertheless, cord blood CD4+ cells are deficient in their ability to provide help for antibody production. Id.
Greater than 90% of cord blood CD4+ cells express high levels of CD45RA and L-selectin (Leu-8) (Id. citing Clement, L T et al, “Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate,” J. Immunol. (1990): 145: 102-108)) and have low levels of CD45RO (citing Sanders et al 1988). Their cytokine profiles suggest that they are naïve THp cells. The dominant immunoregulatory phenotype of cord blood CD4+ cells has been shown to be largely immunosuppressive, consistent with the preponderance of CD4+CD45RA+(and CD38+) cells (Id. citing Tostato, G I et al, “B cell differentiation and immunoregulatory T cell function in human cord blood lymphocytes,” 1980; J. Clin. Invest. 66: 383-880; Jacoby, D R and Oldstone, MBA, “Delineation of suppressor and helper activity within the OKTA4-defined T lymphocyte subset in human newborns,” 1983; J. Immunol. 131: 1765-70; Clement, L T et al, “Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate,” J. Immunol. (1990): 145: 102-108)). Cord blood CD4+ cells cultured with adult B cells and pokeweed mitogen (PWM), or anti CD4+ mAb, demonstrated no helper function (Clement, L T et al, “Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate,” J. Immunol. (1990): 145: 102-108)). However, after activation with phytohemagglutinin (PHA) and culture in IL-2, cord blood CD4++CD45RA+ cells acquired the ability to provide help for B cell differentiation. This functional maturation was accompanied by conversion to the CD4+CD45RA-CD45RO+ phenotype. When the small number of CD4+CD45RA-CD45RO+ cells in cord blood were purified and similarly analyzed, helper activity comparable to that of adult CD4+CD45RA− was found. Id. This helper function was blocked by the presence of even small numbers of cord blood (but not adult) CD4+CD45RA+ cells. Irradiation or mitomycin C treatment of cord blood CD4+CD45RA+ cells abrogated their suppressive activity, but did not induce helper capability. It has been proposed that uncommitted “naïve” CD4+CD45RA+ cells undergo age-related maturational changes that are unrelated to their postulated activation-dependent post-thymic differentiation into CD4+CD45RA− “memory” cells capable of helper functions (Id)
Natural Killer (NK) Cells
Human natural killer (NK) cells can be subdivided into different populations based on the relative expression of the surface markers CD16 and CD56. (Poli, A. et al, “CD56bright natural killer (NK) cells: an important NK cell subset,” Immunol. 2009 April; 126(4): 458-65. Cord blood NK cells are heterogeneous. Although cells bearing the NK marker CD57+ are negligible in cord blood (Cicuttini, F M and Boyd A W, “Hematopoietic and lymphoid progenitor cells in human umbilical cord blood,” Devel. Immunol. 4: 1-11 (1994), citing Abo, T et al, “Post natal expansion of the natural killer and killer cell population in humans identified by the monoclonal HNK-1 antibody,” J. Exp. Med. 1982; 155: 321-26), the proportions of CD16+ lymphocytes are equal to those of adult peripheral blood (Id. citing Tarakkanan, J and Saksela, E, “Umbilical cord blood-derived suppressor cells of the human natural killer cell activity are inhibited by interferon,” Scand. J. Immunol. 1982; 15: 149-57; Perussia, B et al., “Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fcreceptor function. I. Characterization of the lymphocyte subset reactive with B73.1,” J. Immunol. 1983; 130: 2133-41). Spontaneous NK activity of cord blood cells is profoundly reduced compared to the adult. Id. It is thought that CD7+NK+ and CD7+NK− populations may represent a developmental sequence amongst NK cell precursors in human umbilical cord blood, with CD7+NK− cells as candidates for the most immature NK precursor cells in cord blood. Id.
Hematopoietic Stem Cells.
The hematopoietic stem cell is the common ancestor of all blood cells. Hematopoietic stem cell maturation involves the diversification of the lymphoid and myeloid cell lineages, the two major branches of hematopoietic cells. (Kondo, M. “Lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors,” Immunol. Rev. 2010 November; 238(1): 37-46). Lymphoid lineage cells include T, B, and natural killer (NK) cells. The myeloid lineage includes megakaryocytes and erythrocytes (MegE) as well as different subsets of granulocytes (neutrophils, eosinophils and basophils), monocytes, macrophages, and mast cells (GM), which belong to the myeloid lineage (Id. citing Kondo M, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol. 2003; 21:759-806, Weissman I L. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science (New York, N.Y. 2000 Feb. 25; 287(5457):1442-6; see also Iwaskaki, H. and Akashi, K. “Myeloid lineage commitment from the hematopoietic stem cell,”, Immunity 26(6) June 2007, 726-40).
The lymphoid and myeloid lineages are separable at the progenitor level. Common lymphoid progenitors (CLPs) can differentiate into all types of lymphocytes without noticeable myeloid potential under physiological conditions (Kondo M, Scherer D C, Miyamoto T, King A G, Akashi K, Sugamura K, et al. Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature. 2000 Sep. 21; 407(6802):383-6), although some myeloid related genes might be detected in CLPs, depending on the experimental conditions (Delogu A, Schebesta A, Sun Q, Aschenbrenner K, Perlot T, Busslinger M. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity. 2006 March; 24(3):269-81).
Similarly, common myeloid progenitors (CMPs) can give rise to all classes of myeloid cells with no or extensively low levels of B-cell potential (Akashi K, Traver D, Miyamoto T, Weissman I L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000 Mar. 9; 404(6774):193-7). Another cell type, dendritic cells (DCs), is not clearly grouped either in lymphoid or myeloid lineage, because DC can arise from either CLPs or CMPs (Manz M G, Traver D, Miyamoto T, Weissman I L, Akashi K. Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood. 2001 Jun. 1; 97(11):3333-41, Traver D, Akashi K, Manz M, Merad M, Miyamoto T, Engleman E G, et al. Development of CD8alpha-positive dendritic cells from a common myeloid progenitor. Science (New York, N.Y. 2000 Dec. 15; 290(5499):2152-4).
CMPs can proliferate and differentiate into megakaryocyte-erythrocyte (MegE) progenitors and granulocyte-monocyte (GM) progenitors, which further give rise to megakaryocytes, erythrocytes, granulocytes, monocytes and others. (Iwasaki H, Akashi K. Myeloid lineage commitment from the hematopoietic stem cell. Immunity. 2007; 26:726-740).
The monopotent megakaryocyte lineage-committed progenitor (MKPs) has been isolated downstream of MEPs by CD9, a megakaryocyte-associated surface protein. MKPs have the phenotype CD9+IL-7Rα− Lin− Sca-1− c-Kit+Thy1.1− and represent only 0.01% of the total bone-marrow cells. (Iwasaki H, Akashi K. Myeloid lineage commitment from the hematopoietic stem cell. Immunity. 2007; 26:726-740). MKPs give rise exclusively to various sizes of megakaryocyte colonies. (Id. Citing T. N. Nakorn, T. et al, Characterization of mouse clonogenic megakaryocyte progenitors, Proc. Natl. Acad. Sci. USA, 100 (2003), pp. 205-210). MEPs represent the majority of day 8 CFU-S activity; MKPs do not have CFU-S activity, and generate only megakaryocytes in vitro. Id.
Like other primitive hematopoietic cells, bipotent MEPs resemble small lymphocytes but can be distinguished by a specific pattern of cell surface protein display, IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34−/FcRγlo. (Kaushansky, K., “Historical review: megakaryopoiesis and thrombopoiesis,” Blood 2008; 111(3): 981-86). Cells committed to the megakaryocytic lineage then begin to express CD41 and CD61 (integrin αIIbβ3), CD42 (glycoprotein Ib) and glycoprotein V. (Id. Citing Hodohara K, et al., Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK). Blood 2000; 95:769-775; Roth G J, et al., The platelet glycoprotein Ib-V-IX system: regulation of gene expression. Stem Cells 1996; 14 Suppl 1:188-193). Those that are committed to the erythroid lineage begin to express the transferrin receptor (CD71), and as they mature they lose CD41 expression but express the thrombospondin receptor (CD36), glycophorin, and ultimately globin. Id.
The human clonogenic common myeloid progenitors (CMPs) and their downstream progeny, the granulocyte/macrophage (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), reside in the lineage-negative (lin−) CD34+CD38+ fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3Rα chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling. (Manz, M G, et al, “Prospective isolation of human clonogenic common myeloid progenitors,” Proc. Natl Acad. Sci. U.S. 2002 99(18): 11872-11877).
75% of lin−CD34+CD38+IL-3RαloCD45RA+ cells isolated from adult human bone marrow gave rise exclusively to CFU-granulocyte, CFU-macrophage, and CFU-granulocyte/macrophage, whereas 87% of lin−CD34+CD38+IL-3Rα−CD45RA− cells isolated from adult human bone marrow produced burst-forming units erythroid, CFU-megakaryocyte, CFU-megakaryocyte/erythroid, and two CFU-granulocyte (0.5%). In analogy to the defined mouse progenitors the IL-3RαloCD45RA+ cells were termed GMPs and the IL-3Rα−CD45RA− cells termed MEPs. Upon culture on Sys-1 stromal cells with different cytokine combinations, development of both GMPs and MEPs was achieved with SCF, IL-11, FL, Epo, and Tpo after 72 h of culture. IL-3RαloCD45RA− cells gave rise to all types of colonies (but CFU-GEMM), GMPs exclusively gave rise to granulocyte/macrophage colonies, and MEPs gave rise to megakaryocyte/erythrocyte colonies and four (1.6%) granulocyte and macrophage colonies. Therefore, the IL-3RαloCD45RA− cells, which represent the CMP population, can give rise to functional GMPs and MEPs. (Manz, M G, et al, “Prospective isolation of human clonogenic common myeloid progenitors,” Proc. Natl Acad. Sci. U.S. 2002 99(18): 11872-11877).
Gene Expression Profiles.
Whereas granulocyte colony-stimulating factor receptor, C/EBPs, and MPO were expressed by GMPs and not by MEPs, GATA-1, EpoR, c-mpl, β-globin, and von Willebrand factor were detected in MEPs but not in GMPs. None of the myeloid progenitors expressed detectable levels of genes relevant in lymphoid development as TdT, GATA-3, preTα, IL-7Rα, and Pax-5, which were expressed in the lymphoid-committed lin−CD34+CD38+CD10+ progenitors. (Manz, M G, et al, “Prospective isolation of human clonogenic common myeloid progenitors,” Proc. Natl Acad. Sci. U.S. 2002 99(18): 11872-11877).
Myeloid progenitor cells with similar lineage restrictions can be found in cord blood. Although the distinct surface-marker expression profile was similar to adult bone marrow, percentages of the myeloid progenitor populations were slightly different in cord blood: IL-3RαloCD45RA− CMPs account for about 0.4%, IL-3RαloCD45RA+ GMPs for about 0.3%, and IL-3Rα−CD45RA+ MEPs for about 0.05% of the mononuclear cell fraction of umbilical cord blood. HSC-enriched lin−CD34+CD38− cells and CMPs formed all types of colonies with cloning efficiencies of 68 and 83%, respectively. GMPs formed exclusively granulocyte/macrophage colonies (cloning efficiency 41%), and MEPs formed megakaryocyte/erythrocyte colonies (cloning efficiency 88%) with only 4% granulocyte/macrophage colony readout. (Manz, M G, et al, “Prospective isolation of human clonogenic common myeloid progenitors,” Proc. Natl Acad. Sci. U.S. 2002 99(18): 11872-11877).
A subset of HSCs has been shown to express the gene for von Willebrand's factor, a platelet-associated peptide once thought to be restricted to the megakaryocyte lineage. (Smith, B W, and Murphy, G J, “Stem cells, megakaryocytes, and platelets,” Curr. Opin. Hematol. 2014; 21(5): 430-37). These cells produce greater transcript levels of C-mpl, and are primed for megakaryocyte lineage commitment (Id. Citing Sanjuan-Pla, A., et al, “Platelet-biased stem cells reside at the apex of haematopoietic stem-cell hierarchy,” Nature 2013; 502: 232-36). Studies show that transplanted HSCs preferentially home to adjacent megakaryocytes within the endosteal bone marrow niche, in which TPO promotes niche expansion (Id. Citing Olson, T S, et al, “Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radio-ablative conditioning,” Blood 2013; 121: 5238-49) and mature megakaryocytes release cytokines to promote HSC proliferation (Heazlewood, S Y et al, “Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation,” Stem Cell Res. 2013; 11:782-92)). There is also evidence for a myeloid-restricted progenitor that may be a direct descendant of the HSC, completely bypassing the oligopotent progenitor thought to be a crucial intermediary of normal hematopoiesis (Yamamoto, R. et I al, “Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells,” Cell. 2013; 154: 1112-26). This population may descend from CD41+ HSCs, which are more entrenched and less transient than once thought (Gekas, C. and Graf, T., “CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age,” Blood. 2013; 121: 4463-62)).
Transcription Factors
Multiple transcription factors, including Runx1, Gata1, Fli1 and cMyb, form complex networks that regulate the differentiation of megakaryocytes both positively and negatively. (Geddis, A. E., “Megakaryopoiesis,” Semin. Hematol. 2010; 47(3): 212-219). Runx1 interacts with additional megakaryocytic factors including Gata1 and Fli1. Gata1 and its cofactor, Friend of Gata1 (Fog1) are critical in promoting megakaryocyte-erythroid differentiation, while at the same time inhibiting expression of Pu.1 and myeloid differentiation. (Id. Citing Nerlov, C. et al, “GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription,” Blood 2000; 95: 2543-51; Chou S T et al, “Graded repression of PU.1/Sfpil gene transcription by GATA factors regulates hematopoietic cell fate,” Blood 2009; 114: 983-94). Binding sites for Gata1 and Flli1 can be found in the enhancers of many megakaryocyte-specific genes (Id. Citing Eisbacher, M. et al, “Protein-protein interaction between Fli-1 and GATA-1 mediates synergistic expression of megakaryocyte-specific genes through cooperative DNA binding,” Mol. Cell Biol. 2003; 23: 3427-41) and Fli1 enhances the activity of Gata1 at megakaryocytic promoters, and represses the activity of erythroid factors at erythroid promoters. Thus, Fli1 expression may act to restrict the MEP to the megakaryocytic lineage. In contrast, expression of the proto-oncogene c-Myb in the MEM favors erythropoiesis, and c-Myb expression is down regulated during megakaryopoiesis (Metcalf, D et al, “Anamaloous megakaryocytopoiesis in mice with mutations in the c-Myb gene,” Blood 2005; 105: 3480-87).
Megakaryocytes.
Multiple distinct cell population of cord blood-derived megakaryocytes have been observed by flow cytometry, and similar populations were also observed in the megakaryocytic Meg-01 cell line. (Lindsay, C. et al, 2015; Blood 126(23): 4754). The largest cells (called P1), were the most abundant, making up nearly 100% of cells at day 3 in culture. P2 cells, which are smaller and more granular than P1, appeared at day 6 and by day 13 were about 50% of the total. P3 appeared at day 6 and are the smallest, with size and granularity roughly similar to platelets; by day 13 these were about 30% of the total. P1, but not P2 or P3, became CD61/CD41/CD42 positive and CD34 negative over 13 days in culture. 97% and 93% of P2 and P3 cells, respectively, were phosphatidylserine (PS) positive, whereas 93% of P1 cells were PS negative. The PS negative (P1) cells showed many typical features of bone marrow megakaryocytes by electron microscopy, including large size, polypoid nucleus, mitochondria and immature granules, although the demarcation membrane system was poorly developed. Virtually all of the PS positive P2 cells were apoptotic, lacked granules, and had no discernable nuclei. It was found that P1 gives rise to both the P2 and P3 populations, whereas P2 gave rise to no other population. Stimulation of P1, P2 and P3 populations with collagen related peptide, thrombin, protease activated receptor 1-activating peptide (PAR1-AP) and PAR4-AP showed strong integrin activation in P1 cells, but not in P2 or P3 cells. Thus, only a portion of cord blood-derived megakaryocytes are functional.
Growth Factors
Growth factors are extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. These pathways stimulate the accumulation of proteins and other macromolecules, and they do so by both increasing their rate of synthesis and decreasing their rate of degradation.
Platelet α granules contain several different growth factors, including platelet-derived growth factors (PDGF-AA, PDGF-BB, BDGF-AB), transforming growth factor-β (TGF-β1 and TGF-β2), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), and insulin-like growth factor-1 (IGF-1), which are actively secreted by platelets (Aghideh, A N et al, “Platelet growth factors suppress ex vivo expansion and enhance differentiation of umbilical cord blood CD133+ stem cells to megakaryocyte progenitor cells,” Growth Factors 2010; 28(6): 409-16, citing Martieau, I., et al, “Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation,” Biomaterials 2004; 25: 4489-4502). Megakaryocytes express and store platelet factor 4 (PF4), a negative regulator of megakaryopoiesis and hematopoietic stem cell regulation, in alpha granules (Lambert, M P et al, “Intramedullary megakaryocytes internalize released platelet factor 4 and store it in alpha granules,” J. Thromb. Haemost. 2015; 13(10): 1888-99).
Thrombopoietin
Multiple growth factors support megakaryopoesis, the most important of which is megakaryocyte growth and development factor (MGDF), also known as thrombopoietin (TPO). Thrombopoietin, the major regulator of megakaryocyte development and platelet production and a potent stimulator of thrombopoiesis, is a ligand for the Mpl receptor. (Muench, M. and Barcena, A., “Megakaryocyte Growth and Development Factor is a Potent Growth Factor for Primitive Hematopoietic Progenitors in the Human Fetus,” Ped. Res. 2004; 55(6): 1050-56). It can stimulate, both in vitro and in vivo, an increase in megakaryocyte production and megakaryocyte ploidy, and has a broad spectrum of activity on hematopoiesis (Id. Citing Kaushansky, K. “Thrombopoietin: the primary regulator of platelet production,” 1995; Blood 86: 419-311, 2); Kuter, D J et al, 2002 Blood; 100: 3457-69)), and supports the growth of multipotent hematopoietic progenitors and stem cells. (Id.)
TPO belongs to the four-helix bundle family of cytokines, which includes erythropoietin, G-CSF, growth hormone and leukemia inhibitory factor among others. (Geddis, A. E., “Megakaryyopoiesis,” Semin. Hematol. 2010; 47(3): 212-219). The TPO receptor c-Mpl was identified based on its homology to the oncogne v-Mpl, already known at the time as the transforming factor of the murine myeloproliferative leukemia virus (Id. Citing Vigon I, et al. Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor superfamily. Proc Natl Acad Sci USA. 1992; 89:5640-5644). TPO and c-Mpl are critical for megakaryocyte growth and development, and in mouse models where one or the other is absent, platelets and megakaryocytes are reduced to approximately 10% of normal values (Gurney A L, et al. Thrombocytopenia in c-mpl-deficient mice. Science. 1994; 265:1445-1447; Bunting S, et al. Normal platelets and megakaryocytes are produced in vivo in the absence of thrombopoietin. Blood. 1997; 90:3423-3429). In addition to megakaryocytic cells, HSCs also express c-Mpl and depend on TPO signaling for their maintenance and expansion (Id. Citing Fox N, et al, Thrombopoietin expands hematopoietic stem cells after transplantation. J Clin Invest. 2002; 110:389-394).
The c-Mpl gene encodes a 635 amino acid protein consisting of a 25 amino acid signal peptide (1-25), a 465 amino acid extracellular domain (26-491), a 22 residue transmembrane domain (492-513) and an intracellular domain containing two conserved motifs termed box 1 (528-536) and box 2 (565-574). The extracellular domain is composed of two repeating modules; the membrane distal module appears to have an inhibitory effect on signaling, as its deletion results in constitutive activation of the receptor (Id. Citing Sabath D F, Kaushansky K, Broudy V C. Deletion of the extracellular membrane-distal cytokine receptor homology module of Mpl results in constitutive cell growth and loss of thrombopoietin binding. Blood. 1999; 94:365-367). c-Mpl does not have intrinsic kinase activity, but instead associates with the cytoplasmic tyrosine kinase Janus kinase 2 (Jak2) through its box 1 domain (Id. Citing Drachman J G, Kaushansky K. Structure and function of the cytokine receptor superfamily. Curr Opin Hematol. 1995; 2:22-28). Additional elements regulate receptor internalization and subsequent degradation following TPO binding. These include dileucine repeats located within box 2, Tyr591 and Tyr625 (Id. Citing Dahlen D D, et al., Internalization of the thrombopoietin receptor is regulated by 2 cytoplasmic motifs. Blood. 2003; 102:102-108; Hitchcock I S, et al, YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation. Blood. 2008).
TPO signaling depends on the activation of Jak2. Jak2 associates with box 1 of c-Mpl through its FERM (band 4.1/ezrin/radixin/moesin) domain. Based on X-ray crystal studies of the erythropoietin receptor (Id. Citing Livnah O, et al, Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science. 1999; 283:987-990), it is believed that in the unliganded state c-Mpl exists as a homodimer, and that TPO binding results in a conformational change that brings the cytoplasmic tails of the receptor into closer proximity. Consequently, the Jak2 molecules associated with the receptor are brought close enough to each other to become activated through trans-autophosphorylation (Id. Citing Witthuhn B A, Quelle F W, Silvennoinen O, Yi T, Tang B, Miura O, et al. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell. 1993; 74:227-236). Active Jak2 then phosphorylates itself on multiple residues and phosphorylates c-Mpl on at least Tyr625 and Tyr630 (Id. Citing Drachman J G, Kaushansky K. Dissecting the thrombopoietin receptor: functional elements of the Mpl cytoplasmic domain. Proc Natl Acad Sci USA. 1997; 94:2350-2355). These phosphotyrosine residues provide docking sites for src homology 2 (SH2)-domain-containing signaling proteins that modulate receptor signaling.
Following the activation of Jak2, multiple signaling molecules are activated and mediate the cellular response to TPO. These include members of the signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol-3 kinase (PI3K) pathways (Id. Citing Geddis A E, et al, Thrombopoietin: a pan-hematopoietic cytokine. Cytokine Growth Factor Rev. 2002; 13:61-73). Jak2 directly phosphorylates STAT family members including STAT1, 3, 5a and 5b (Id. Citing Schulze H, et al., Thrombopoietin induces the generation of distinct Stat1, Stat3, Stat5a and Stat5b homo- and heterodimeric complexes with different kinetics in human platelets. Exp Hematol. 2000; 28:294-304). Once phosphorylated, these STAT proteins dimerize and translocate to the nucleus of the cell where they can bind to STAT-responsive transcriptional elements within genes such as p21 (Id. Citing Matsumura I, et al. Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21(WAF1/Cip1) by STAT5. Mol Cell Biol. 1997; 17:2933-2943), Bcl-xL (Id. Citing Kirito K, et al, Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways. J Biol Chem. 2002; 277:8329-8337) and cyclin D1 (Id. Citing Magne S, et al, STAT5 and Oct-1 form a stable complex that modulates cyclin D1 expression. Mol Cell Biol. 2003; 23:8934-8945). Constitutive activation of the Jak2/STAT pathway can lead to cytokine-independent growth and contribute to transformation, as demonstrated by the finding of mutant Jak2 in myeloproliferative disorders, translocations involving Jak2 in lymphoid leukemias, and constitutively active STAT5 in leukemic cell lines (Id. Citing Harir N, et al. Constitutive activation of Stat5 promotes its cytoplasmic localization and association with PI3-kinase in myeloid leukemias. Blood. 2007; 109:1678-1686; Najfeld V, et al, Numerical gain and structural rearrangements of JAK2, identified by FISH, characterize both JAK2617V>F-positive and -negative patients with Ph-negative MPD, myelodysplasia, and B-lymphoid neoplasms. Exp Hematol. 2007; 35:1668-1676).
Jak2 also activates the small GTPase Ras and the MAPK cascade, culminating in the activation of extracellular signal-related kinase (ERK)1/2. Multiple studies have demonstrated the importance of TPO-induced MAPK signaling in megakaryocytic differentiation (Id. Citing Rouyez M C, et al, Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway. Mol Cell Biol. 1997; 17:4991-5000; Rojnuckarin P, et al, Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis. Blood. 1999; 94:1273-1282; Fichelson S, et al. Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors. Blood. 1999; 94:1601-1613). The classical pathway by which TPO signaling is thought to activate Ras depends on the binding of the adaptor protein Shc to phosphorylated c-Mpl Tyr625 (Id. Citing Drachman J G, Kaushansky K. Dissecting the thrombopoietin receptor: functional elements of the Mpl cytoplasmic domain. Proc Natl Acad Sci USA. 1997; 94:2350-2355; Miyakawa Y, et al. Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets. Blood. 1995; 86:23-27) and the assembly of a complex containing the adaptor protein Grb2 and the guanine nucleotide exchange factor SOS (Id. Citing Alexander W S, et al, Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation. Embo J. 1996; 15:6531-6540; Skolnik E Y, et al. The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science. 1993; 260:1953-1955). Ras then activates Raf-1, mitogen-induced extracellular kinase (MEK) and finally Erk 1/2 (Id. Citing Avruch J, et al. Ras activation of the Raf kinase: tyrosine kinase recruitment of the MAP kinase cascade. Recent Prog Horm Res. 2001; 56:127-155). Although activation of MAPK is significantly reduced in the absence of c-Mpl Tyr625 and Tyr630, it is not eliminated (Id. Citing Luoh S M, et al. Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo. Mol Cell Biol. 2000; 20:507-515), suggesting that activation of Erk1/2 can be mediated either through a Shc-independent mechanism, possibly through Grb2/Sos complexes recruited to Jak2 (Id. Citing Brizzi M F, et al, Discrete protein interactions with the Grb2/c-Cbl complex in SCF- and TPO-mediated myeloid cell proliferation. Oncogene. 1996; 13:2067-2076). Alternatively, the small GTPase Rap1 can activate Erk1/2 via B-Raf independent of Ras (Id. Citing Garcia J, et al, Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1- and Rap1-B-Raf-dependent pathways. Mol Cell Biol. 2001; 21:2659-2670).
The PI3K pathway is also essential for megakaryopoiesis (Id. Citing Geddis A E, Fox N E, Kaushansky K. Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors. J Biol Chem. 2001; 276:34473-34479). PI3K is composed of a kinase (p110) and a regulatory subunit (p85). TPO induces formation of a complex between phosphorylated p85 and the adaptor Gab, although this complex has not been found to bind directly to c-Mpl (Id. Citing Miyakawa Y, et al, Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes. J Biol Chem. 2001; 276:2494-2502); alternatively, PI3K may become activated indirectly through Ras (Id. Citing Kodaki T, et al, The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol. 1994; 4:798-806). TPO-induced PI3K phosphorylates and activates the serine/threonine kinase Akt whose substrates include Forkhead, glycogen synthase kinase 3 beta (GSK-3β) and Bad (Id. Citing Geddis A E, Fox N E, Kaushansky K. Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors. J Biol Chem. 2001; 276:34473-34479; Nakao T, et al, PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27(Kip1) Cell Cycle. 2007; 7; Soda M, et al, Inhibition of GSK-3beta promotes survival and proliferation of megakaryocytic cells through a beta-catenin-independent pathway. Cell Signal. 2008; 20:2317-2323), collectively promoting survival and proliferation of megakaryocytic cells. PI3K also activates mammalian target of rapamycin (mTOR), whose targets SK6 and 4E-BP1 increase proliferation and maturation of megakaryocytic progenitors (Id. Citing Raslova H, et al. Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation. Blood. 2006; 107:2303-2310; Guerriero R, et al. Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes. J Cell Sci. 2006; 119:744-752). PI3K is itself negatively regulated by phosphatase and tensin homolog (PTEN), a tumor suppressor that promotes quiescence in hematopoietic stem cells (HSC) (Id. Citing Zhang J, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature. 2006; 441:518-522). Although PTEN regulates the activity of Akt and mTOR, its role in TPO signaling and megakaryopoiesis has not yet been defined.
Checks on TPO signaling and megakaryopoiesis are required to maintain homeostatic balance. To ensure that signals are appropriately terminated, many positive regulators also induce their own inhibitors. For example, activation of Jak/STAT pathway induces the transcription of members of the suppressor of cytokine signaling (SOCS) family (Id. Citing Starr R, et al. A family of cytokine-inducible inhibitors of signalling. Nature. 1997; 387:917-921; Endo T A, et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature. 1997; 387:921-924). This family includes at least 8 members that can inhibit Jak signaling in a variety of ways, including binding to the activation loop of Jak and targeting it for degradation, acting as a pseudosubstrate for Jak, or binding to phosphorylated tyrosines within the cytokine receptor itself (Id. Citing Alexander W S, Hilton D J. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol. 2004; 22:503-529). Induction of a SOCS response from one receptor can negatively regulate another, thereby providing a mechanism for cytokine cross-talk; this is illustrated by the finding that treatment of megakaryocytes with interferon-α induces SOCS1, which then down-regulates TPO signaling through inhibition of Jak2 (Id. Citing Wang Q, Miyakawa Y, Fox N, Kaushansky K. Interferon-alpha directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1. Blood. 2000; 96:2093-2099).
Jak2 has other binding partners that regulate its activity. For example, Lnk is an adaptor protein that inhibits growth in HSCs, erythroid and megakaryocytic cells (Id. Citing Tong W, Lodish H F. Lnk inhibits Tpo-mpl signaling and Tpo-mediated megakaryocytopoiesis. J Exp Med. 2004; 200:569-580; Seita J, et al. Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction. Proc Natl Acad Sci USA. 2007; 104:2349-2354). Lnk binds to phosphorylated tyrosines within Jak2 through its SH2-domain (Id. Citing Bersenev A, et al, Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2. J Clin Invest. 2008; 118:2832-2844); however, the exact mechanism by which it inhibits TPO signaling is not understood. In addition to binding negative regulators, Jak2 may be phosphorylated within the FERM domain, inducing its dissociation from c-Mpl and thus providing another mechanism to ‘turn off’ signaling (Id. Citing Funakoshi-Tago M, et al, Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2. Embo J. 2006; 25:4763-4772).
Some extracellular signal proteins, including platelet-derived growth factor (PDGF), can act as both growth factors and mitogens, stimulating both cell growth and cell-cycle progression. This functional overlap is achieved in part by overlaps in the intracellular signaling pathways that control these two processes. The signaling protein Ras, for example, is activated by both growth factors and mitogens. It can stimulate the PI3-kinase pathway to promote cell growth and the MAP-kinase pathway to trigger cell-cycle progression. Similarly, Myc stimulates both cell growth and cell-cycle progression. Extracellular factors that act as both growth factors and mitogens help ensure that cells maintain their appropriate size as they proliferate.
Since many mitogens, growth factors, and survival factors are positive regulators of cell-cycle progression, cell growth, and cell survival, they tend to increase the size of organs and organisms. In some tissues, however, cell and tissue size also is influenced by inhibitory extracellular signal proteins that oppose the positive regulators and thereby inhibit organ growth. The best-understood inhibitory signal proteins are TGF-β and its relatives. TGF-β inhibits the proliferation of several cell types, either by blocking cell-cycle progression in G1 or by stimulating apoptosis. TGF-β binds to cell-surface receptors and initiates an intracellular signaling pathway that leads to changes in the activities of gene regulatory proteins called Smads. This results in complex changes in the transcription of genes encoding regulators of cell division and cell death.
Bone morphogenetic protein (BMP), a TGF-β family member, helps trigger the apoptosis that removes the tissue between the developing digits in the mouse paw. Like TGF-β, BMP stimulates changes in the transcription of genes that regulate cell death.
Fibroblast Growth Factor (FGF)
The fibroblast growth factor (FGF) family currently has over a dozen structurally related members. FGF1 is also known as acidic FGF; FGF2 is sometimes called basic FGF (bFGF); and FGF7 sometimes goes by the name keratinocyte growth factor. Over a dozen distinct FGF genes are known in vertebrates; they can generate hundreds of protein isoforms by varying their RNA splicing or initiation codons in different tissues. FGFs can activate a set of receptor tyrosine kinases called the fibroblast growth factor receptors (FGFRs). Receptor tyrosine kinases are proteins that extend through the cell membrane. The portion of the protein that binds the paracrine factor is on the extracellular side, while a dormant tyrosine kinase (i.e., a protein that can phosphorylate another protein by splitting ATP) is on the intracellular side. When the FGF receptor binds an FGF (and only when it binds an FGF), the dormant kinase is activated, and phosphorylates certain proteins within the responding cell, activating those proteins.
FGFs are associated with several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and axon extension. While FGFs often can substitute for one another, their expression patterns give them separate functions. FGF2 is especially important in angiogenesis, whereas FGF8 is involved in the development of the midbrain and limbs.
The expression levels of angiogenic factors, such as VEGF, IGF, PDGF, HGF, FGF, TGFm Angiopoeitin-1, and stem cell factor (SCF) have been found to differ amongst bone-derived-, cartilage-derived-, and adipose-derived MSCs. (Peng et al., 2008, Stems Cells and Development, 17: 761-774).
Insulin-Like Growth Factor (IGF-1)
IGF-1, a hormone similar in molecular structure to insulin, has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cells, and lungs. It plays an important role in childhood growth and continues to have anabolic effects in adults. IGF-1 is produced primarily by the liver as an endocrine hormone as well as in target tissues in a paracrine/autocrine fashion. Production is stimulated by growth hormone (GH) and can be retarded by undernutrition, growth hormone insensitivity, lack of growth hormone receptors, or failures of the downstream signaling molecules, including SHP2 and STAT5B. Its primary action is mediated by binding to its specific receptor, the Insulin-like growth factor 1 receptor (IGF1R), present on many cell types in many tissues. Binding to the IGF1R, a receptor tyrosine kinase, initiates intracellular signaling; IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. IGF-1 is a primary mediator of the effects of growth hormone (GH). Growth hormone is made in the pituitary gland, released into the blood stream, and then stimulates the liver to produce IGF-1. IGF-1 then stimulates systemic body growth. In addition to its insulin-like effects, IGF-1 also can regulate cell growth and development, especially in nerve cells, as well as cellular DNA synthesis.
Transforming Growth Factor Beta (TGF-β)
There are over 30 structurally related members of the TGF-β superfamily, and they regulate some of the most important interactions in development. The proteins encoded by TGF-β superfamily genes are processed such that the carboxy-terminal region contains the mature peptide. These peptides are dimerized into homodimers (with themselves) or heterodimers (with other TGF-β peptides) and are secreted from the cell. The TGF-β superfamily includes the TGF-β family, the activin family, the bone morphogenetic proteins (BMPs), the Vg-1 family, and other proteins, including glial-derived neurotrophic factor (GDNF, necessary for kidney and enteric neuron differentiation) and Millerian inhibitory factor, which is involved in mammalian sex determination. TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating the formation of the extracellular matrix between cells and for regulating cell division (both positively and negatively). TGF-β1 increases the amount of extracellular matrix epithelial cells make both by stimulating collagen and fibronectin synthesis and by inhibiting matrix degradation. TGF-βs may be critical in controlling where and when epithelia can branch to form the ducts of kidneys, lungs, and salivary glands.
Among various hematoregulatory cytokines examined, TGF-β1 was by far the most potent enhancer of mRNA expression of bone marrow stromal TPO, a commitment of lineage specificity. The TPO, in turn, induced TGB-β receptors I and II on megakaryoblasts at the midmegakaryopoietic stage. At this stage, TGF-β1 was able to arrest the maturation of megakaryocyte colony forming units (CFU-Meg) in a dose-dependent manner. This effect was relatively specific when compard with its effect on burst-forming unit-erythroid (BFU-E) or CFU-GM. (Sakamaki, S. et al, “Transforming growth factor-31 (TGF-β1) induces thrombopoietin from bone marrow stromal cells, which stimulates the expression of TGF-β receptor on megakaryocytes and, in turn, renders them susceptible to suppression by TGF-β itself with high specificity,” Blood 1999; 94: 1961-70).
Activin A and BMP 2 induce cell commitment and differentiation toward erythropoiesis, even in the absence of erythropoietin (EPO). Their biological activities are antagonized by binding with follistatin or FLRG (follistatin-related gene), 2 secreted glycoproteins expressed by human bone marrow and regulated by TGF-β and activin A ((Jeanpierre, S. et al, “BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling,” Blood 2008; 112: 3154-63) citing Maguer-Satta V, et al., Regulation of human erythropoiesis by activin A, BMP2, and BMP4, members of the TGFbeta family. Exp Cell Res 2003; 282:110-120; Maguer-Satta V, Rimokh R., FLRG, member of the follistatin family, a new player in hematopoiesis. Mol Cell Endocrinol 2004; 225:109-118). FLRG and follistatin are involved in the regulation of human hematopoietic cell dhesiveness in immature hematopoietic progenitors and stem cells through direct interactions between the type I motifs of fibronectin and follistatin domains. (Id. Citing Maguer-Satta V, et al., A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin. Exp Cell Res 2006; 312:434-442 10).
Bone Morphogenetic Proteins (BMPs)
The members of the BMP family were originally discovered by their ability to induce bone formation. Bone formation, however, is only one of their many functions, and they have been found to regulate cell division, apoptosis (programmed cell death), cell migration, and differentiation. BMPs can be distinguished from other members of the TGF-β superfamily by their having seven, rather than nine, conserved cysteines in the mature polypeptide. The BMPs include proteins such as Nodal (responsible for left-right axis formation) and BMP4 (important in neural tube polarity, eye development, and cell death).
In humans, BMP2, BMP4 and BMP7 regulate the proliferation, maintenance (Jeanpierre, S. et al, “BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling,” Blood 2008; 112: 3154-63) citing Hutton, J F, et al, “Bone morphogenetic protein 4 contributes to the maintenance of primitive cord blood hematopoietic progenitors in an ex vivo stroma-non contact co-culture system,” Stem Cell Dev. 2006; 15: 805-13), clonogenicity, and repopulating capacity of CD34+CD38− primitive hematopoietic populations (Id. Citing Bhatia, M. et al, “Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells,” J. Exp. Med. 1999; 189: 1139-48). BMP2 and BMP4, either alone or in combination with activin A, have been shown to regulate erythropoiesis in various models (Id. Citing Maguer-Satta, V, and Rimokh, R, “FLRG, member of the follistatin family, a new player in hematopoiesis,” Mol. Cell Endocrinol. 2004; 225: 109-11).
It has been shown that BMP4 cooperates with SCF to modulate the primitive hematopoietic stem cell compartment in the absence of any other cytokine. Id.
Of the TGFβ family, only BMP4 has the same capacity as TPO to induce early and late MK markers, and similar terminal differentiation properties, such as polyploidization, secretion of PF4, and platelet production. (Id). It has been demonstrated that BMP4, an element of a key signaling pathway involved in the regulation of the hematopoietic “niche” (Id. Citing Zhang, et al, “Identification of the haematopoietic stem cell niche and control of the niche size,” Nature 2003; 425: 836-41), which is mainly produced by the bone marrow stroma (Id. Citing Martinovic, S. et al, “Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture,” J. Histochem. Cytochem. 2004; 52: 1159-67), localized in human megakaryocytes and platelets (Id. Citing Sipe, J B et al, “Localization of bone morphogenetic proteins (BMPs)-2, -4, and -6 within megakaryocytes and platelets,” Bone 2004; 35: 1316-22) and autologously produced by MK progenitors, efficiently regulates all stages of human megakaryopoiesis, from maintenance of primitive uncommitted progenitors to late stages of MK differentiation. Furthermore, data suggest that the JAK/STAT and mTOR signaling pathways are involved in the regulation of MK maturation by BMP4, as confirmed for TPO (Id. Citing Guerriero, R. et. Al., “Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes,” J. Cell Sci. 2006; 119: 744-52, Raslova, H et al, “Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation,” Blood 2006; 107: 2303-10). The reported results thus indicate that BMP4 and TPO use similar signaling pathways to regulate human MK differentiation. Id. Moreover, using specific extracellular inhibitors of TPO or BMP4, it was shown that whereas either inhibitor of the BMP4 signaling pathway efficiently inhibited the effects of TPO, anti-TPO-R antibodies were not able to block the effects of BMP4 on MK differentiation. Id. Moreover, TPO induced BMP4 synthesis and BMP receptor expression in MK progenitors, suggesting that whereas TPO uses the BMP4 signaling pathway to regulate human MK, the reverse does not seem to be true. Id.
PEAR-1, RAD001, Wnt3a, and AHR
Other factors implicated as regulators of megakaryopoiesis include platelet endothelial aggregation receptor-1 (PEAR-1), a stimulator of PI3K/PTEN signaling (Smith, B W, and Murphy, G J, “Stem cells, megakaryocytes, and platelets,” Curr. Opin. Hematol. 2014; 21(5): 430-37); citing Kauskot, A. et al, “PEAR1 attenuates megakaryopoiesis via control of the PI3K/PTEN pathway,” Blood. 2013; 121: 5208-17) and RAD001, an mTOR inhibitor (Id. Citing Su-Y-C et al, “RAD001-mediated STAT3 upregulation and megakaryocytic differentiation,” Thromb. Haemost. 2013; 109: 540-49)). Wnt3a has been implicated as a repressor of human megakaryocyte progenitor expansion in an in-vitro iPSC derivation system that causes production of CD41/CD235 dual positive progenitors (Id. Citing Paluru, P. et al, “The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from hyman embryonic stem cells,” Stem Cell Res. 2014; 12: 441-51). A role for the aryl hydrocarbon receptor (AHR) in the regulation of iPSC-based in vitro megakaryopoiesis (Id. Citing Smith, B W et al, “The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation,” Blood. 2013; 122: 376-85) has been described.
Platelet Derived Microparticles and Exosomes
Platelets have a well-described physiological role in hemostasis and coagulation, but recently, they have also been shown to participate in immunity, tissue repair and development (Elzey B D, et al., The emerging role of platelets in adaptive immunity. Cell Immunol. 2005; 238:1-9; Jenne C N et al., Platelets: bridging hemostasis, inflammation, and immunity. Int J Lab Hematol. 2013; 35:254-61; Bertozzi C C et al., Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling. Blood. 2010; 116:661-70). Platelet-derived extracellular vesicles (EVs) can provide the molecules necessary to orchestrate these diverse functions. Platelets can generate microvesicles or microparticles (MPs), which are derived from the plasma membrane, and exosomes (EXOs), which are derived from endosomal pathways (Aatonen M T et al., Isolation and characterization of platelet-derived extracellular vesicles, J. Extracellular Vesicles, vol. 3 (2014) 24692).
Platelet plasma membrane derived microparticles (PMPs) are generally known to be 100 to 1000 nm in size. Platelets are also known to produce exosomes, which are 40 to 100 nm in size, from multivesicular bodies. In contrast to the heterogenous PMPs, exosomes in general form a more homogenous population, both by size and molecular content, but in platelets, the normally distinct formation processes of the two are jumbled because of α-granules. Multivesicular bodies, the source of exosomes, are also considered to be pre stages of α-granules, which may then liberate exosomes on fusion with the plasma membrane, and several α-granule-derived molecules are also present on PMPs. The molecular markers present on or in platelet-derived microvesicles, plasma membrane-derived microparticles, and platelet-derived exosomes include, without limitation, the following: Growth factors such as VEGF, bFGF, PDGF, TGF-beta1; Immune response factors such as CD40L(CD154); Chemokines/cytokines such as Rantes(CCL5), CCL23, CXCL7, CXCR4, PF-4(CXCL4), TNF-RI-II, IL-1 beta, CX3CR1, and beta-thromboglobulin; Complement proteins such as CD55, CD59, C5b-9, C1q, C3B, C1-INH, Factor H; Apoptosis markers such as Caspace-3, Caspace-9, FasR(CD95); Coagulation factors such as Fva, FVIII, TFPI, TF, PAR-1, FXIIIA; Active Enzymes such as PDI, 12-LO, NADPH oxidase, iNOS2, Heparnase; Adhesion proteins such as alpha-IIb/beta3 (CD41/CD61), GPIb (CD42b), GPIX (CD42a), P-selectin (CD62P), PECAM-1 (CD31), GPIIIb (CD36), CD49, CD29, CD47, CD9, JAM-A, vWF, fibrinogen, thrombospondin, vitronectin; Bioactive lipids such as PS, AA, LPA, TXA2; among other miscellaneous markers such as Peta-3 (CD151), CD63, PPAR-gamma, TIMP3, Lactadherin, PAI-1, PrPC, beta2GPI (Aatonen et al., Seminars in Thrombosis and Hemostasis Vol. 38, No. 1 (2012)). The common exosomal marker, CD63, is not only enriched in the platelet-derived exosomes but is also present on PMPs and, vice versa, many common PMP proteins are detected on subsets of exosomes.
Platelet-derived microvesicles (PMVs) seem to participate in diverse and sometimes paradoxal activities such as coagulation, adhesion, inflammation, immunity, and apoptosis. In many of these homeostatic activities, several cell types work in concert and may provide microvesicles (MVs) for intercellular communication. This dialogue depends on the formation of functionally variable MVs tailored for the purpose. The effect of PMVs can be either direct, that is, mediated by the PMV itself, such as acting as a catalytic surface, or indirect, that is, mediated by the recipient cells, which change their phenotype on PMV fusion. However, the presence of a molecule is not a guarantee for its function, as demonstrated by the unexpected anti-inflammatory response induced by CD40L(CD154)-containing PMPs (Aatonen et al., Seminars in Thrombosis and Hemostasis Vol. 38, No. 1 (2012)). The ultimate effect of PMVs is likely to depend on the cellular milieu (both temporally and spatially), which may explain, for example, the apparently contradictory pro- and anticoagulant capacity of the same PMVs.
PMVs can transfer fully operational surface receptors (CXC4R, CD41) onto the recipient cells. Receptor transfer by PMVs may confound the origin of cells: PMP-mediated transfer of CD31 and von Willebrand factor into monocytes falsely implied a presence of endothelial progenitor cells. PMVs also contain and transfer active enzymes, for example protein disulfide isomerase for platelet aggregation, inducible nitric oxide synthase II and nicotinamide adenine dinucleotide phosphate oxidase during endothelial dysfunction, and 12-lipo-oxygenase in lipoxin A4 production from mast cells. The participation of PMVs in innate and adaptive immunity is further inferred by the presence of several molecule groups such as cytokines and chemokines and their receptors, CD40L, and PF4 (Aatonen et al., Seminars in Thrombosis and Hemostasis Vol. 38, No. 1 (2012)).
Platelets harbor RNA molecules which are translated into proteins in an activation-dependent manner, for example CD41, CD61, and IL-1β33 which are all members of the PMP proteome. It has been suggested that agonist-dependent changes in the platelet translatome may underlie the molecular, or even the functional, differences of PMP species (Aatonen et al., Seminars in Thrombosis and Hemostasis Vol. 38, No. 1 (2012)).
Previous Work Using Adult Peripheral Blood Platelets
In our previous work, we identified embryonic-like stem cells isolated from adult human peripheral blood, designated as peripheral blood-stem cells (PB-SC), which display characteristics of pluripotent cells. These cells were shown to have the capability of proliferation and differentiation into other cell types making them suitable for use in stem cell based therapies. These cells, which display embryonic stem cell characteristics and hematopoietic cell characteristics, are phenotypically distinct from lymphocytes, macrophages, monocytes, and hematopoietic stem cells.
The described invention provides umbilical cord blood derived platelet-like cells that can be used to generate induced pluripotent stem cells from adult mononuclear cells without the safety concerns involved in the generation of induced pluripotent stem cells by viral- or drug-induced transduction that may be stable when transferred to the patient.